A peptide family which has been the focus of much research, and efforts to improve its administration and bioavailability, is the hedgehog family of proteins. The hedgehog proteins are a family of extracellular signaling proteins that regulate various aspects of embryonic development both in vertebrates and in invertebrates (for reviews see Perrimon, N. (1995) Cell 80, 517-520 and Johnson, R. L., and Tabin, C. (1995) Cell 81, 313-316). The most well-characterized hedgehog protein is Sonic hedgehog (Shh), involved in anterior-posterior patterning, formation of an apical ectodermal ridge, hindgut mesoderm, spinal column, distal limb, rib development, and lung development, and in inducing ventral cell types in the spinal cord, hindbrain and forebrain (see Riddle, R. D., et al. (1993) Cell 75, 1401-1416; Echelard, Y. et al. (1993) Cell 75, 1417-1471; Roelink, H., et al. (1994) Cell 76, 761-775; and Roelink, H., et al. (1995) Cell 81, 445-455).
While the mechanism of action of hedgehog proteins is not understood fully, the most recent biochemical and genetic data suggest that the receptor for Shh is the product of the tumor suppressor gene, patched (Marigo, V., et al. (1996) Nature 384, 176-179; Stone, D. M., et al. (1996) Nature 384, 129-134) and that other proteins; smoothened (Alcedo, J., et al. (1996) Cell 86, 221-232), Cubitus interruptus or its mammalian counterpart gli (Dominguez, M., et al. (1996) Science 272, 1621-1625; Alexandre, C., et al. (1996) Genes & Dev. 10, 2003-2013), and fused (Therond, P. P., et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4224-4228) are involved in the hedgehog signaling pathway.
Human Shh is synthesized as a 45 kDa precursor protein that is cleaved autocatalytically to yield: (I) a 20 kDa N-terminal fragment that is responsible for all known hedgehog signaling activity (SEQ ID NOS. 6 and 24); and (II) a 25 kDa C-terminal fragment that contains the autoprocessing activity (Lee, J. J., et al. (1994) Science 266, 1528-1536; Bumcrot; D. A., et al. (1995) Mol. Cell Biol. 15, 2294-2303; Porter, J. A., et al. (1995) Nature 374, 363-366). The N-terminal fragment of naturally occurring hedgehog consists of amino acid residues 24-197 of the full-length precursor sequence, of which the N-terminal amino acid residue is a cysteine.
The N-terminal fragment remains membrane-associated through the addition of a cholesterol at its C-terminus (Porter, J. A., et al. (1996) Science 274, 255-258; Porter, J. A., et al. (1995) Cell 86, 21-34) and a fatty acid at its N-terminus (Pepinsky et al., (1998) J. Biol. Chem. 273, 14037-14045). These modifications are critical for restricting the tissue localization of the hedgehog signal. The addition of the cholesterol is catalyzed by the C-terminal domain during the processing step.
A major factor limiting the usefulness of proteinaceous substances such as hedgehog for their intended application is that, when given parenterally, they are eliminated from the body within a short time. This can occur as a result of metabolism by proteases or by clearance using normal pathways for protein elimination such as by filtration in the kidneys. The oral route of administration of these substances is even more problematic because in addition to proteolysis in the stomach, the high acidity of the stomach may inactivate them before they reach their intended target tissue. The problems associated with these routes of administration of proteins are well known in the pharmaceutical industry, and various strategies are being used in attempts to solve them.
A great deal of work dealing with protein stabilization has been published. One method of stabilization that has been widely used is the addition of an inert polymer to the protein. Numerous ways of conjugating selected amino acid residues of proteins (e.g., cysteines, lysines, N-terminal residues) with polymeric materials are known, including use of dextrans, polyvinyl pyrrolidones, glycopeptides, polyethylene glycol and polyamino acids. The resulting conjugated polypeptides are reported to retain their biological activities and solubility in water for parenteral applications.
In the case of hedgehog, we have previously discovered that in certain cell types, the protein undergoes proteolytic clipping at various sites in the N-terminal domain. Moreover, these N-terminally clipped forms of hedgehog are inactive in the 10T1/2 assay (in which the cell line 10T1/2 exhibits upregulation of Alkaline phosphatase when cultured for five days in the presence of active Sonic Hedgehog protein). In particular, sonic hedgehog lacking the first 10 amino acids of its N-terminus is inactive and also antagonizes wild-type SHH when both forms are present in the assay. (U.S. Ser. No. 60/106,703). Thus, if one wants to produce a fully active protein that can be further stabilized with a non-hedgehog moiety such as a polymer, one needs to prevent N-terminal proteolytic clipping.